New Insights into the Interaction of Ras with the Plasma Membrane

Magee, Tony; Marshall, Chris

doi:10.1016/s0092-8674(00)80601-7

Cited by 149 publications

(97 citation statements)

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“…1 Ras and Ras-related proteins depend on the covalent attachment of these isoprenoids to their C-terminus for their proper localization to the cell membrane. 2 Plasma membrane localization of Ras is necessary for the conversion of its inactive GDP-to its active GTPbound state, 49 following activation of transmembrane recep- tors by growth factors such as the myeloma growth and survival factor IL-6. 22-24 GTP-bound Ras subsequently activates downstream signaling proteins, which regulate growth and survival.…”

Section: Discussionmentioning

confidence: 99%

“…1 These isoprenoids are used for post-translational modification of a variety of proteins, including Ras and Ras-related GTP binding proteins. Isoprenylation is essential for membrane attachment 2 and participation of the modified proteins in signal transduction processes. [3][4][5][6][7] Lovastatin is a nonreversible competitive inhibitor of HMGCoA reductase and is widely used for the treatment of hypercholesterolemia.…”

Section: Introductionmentioning

confidence: 99%

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The cholesterol lowering drug lovastatin induces cell death in myeloma plasma cells

et al. 2002

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Lovastatin is an irreversible inhibitor of HMG-CoA reductase and blocks the production of mevalonate, a critical compound in the production of cholesterol and isoprenoids. Isoprenylation of target proteins, like the GTP-binding protein Ras, is essential for their membrane localization and subsequent participation in intracellular signaling cascades. Lovastatin effectively decreased the viability of plasma cells from cell lines (n = 10) and myeloma patients' samples (n = 8) in a dose-and time-dependent way. Importantly, co-incubation of lovastatin with dexamethasone had a synergistic effect in inducing plasma cell cytotoxity. This effect was not the consequence of a change in the protein expression levels of Bcl-2 or Bax induced by lovastatin. The decrease in plasma cell viability was the result of induction of apoptosis and inhibition of proliferation. Mevalonate effectively reversed the cytotoxic and cytostatic effects of lovastatin in plasma cells. The cytotoxic activity of lovastatin was higher in Pgp expressing cell lines, but did not correlate with the multidrug resistance (MDR)-related proteins LRP, Bcl-2 and Bax. Lovastatin treatment resulted in a shift of Ras localization from the membrane to the cytosol that was reversed by mevalonate. The data presented in this paper warrant study of lovastatin alone or in combination with therapeutic drugs, in the treatment of myeloma patients.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The cholesterol lowering drug lovastatin induces cell death in myeloma plasma cells

et al. 2002

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“…Their association with membranes is dependent on the cotranslational and posttranslational covalent attachment of fatty acids (Milligan et al, 1995;Wedegaertner et al, 1995;Bhatnagar and Gordon, 1997). Although membrane association is crucial for their function, little is known of the mechanisms by which these proteins are targeted to specific membrane compartments (Magee and Marshall, 1999). To obtain insight into this, we followed previously the fate of newly synthesized Lck, a Src family protein, by pulse-chase labeling experiments.…”

Section: Discussionmentioning

confidence: 99%

Hsp90 Is Essential for the Synthesis and Subsequent Membrane Association, But Not the Maintenance, of the Src-Kinase p56^lck

Bijlmakers

Marsh

2000

MBoC

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Tyrosine kinases of the Src family are synthesized as cytosolic proteins that subsequently translocate to membranes. Little is known of the mechanisms responsible for targeting these proteins to membranes, although a role for the cytosolic chaperone Hsp90 has been proposed. Here, we have studied the involvement of Hsp90 in the synthesis, membrane binding, and maintenance of the Src-kinase Lck. Using specific inhibitors of Hsp90, geldanamycin and radicicol, we found that functional Hsp90 is essential for the stability of newly synthesized, but not mature, Lck. Similar results were obtained for two other Src-kinases, c-Src and Lyn. In contrast, LckY505F and LckΔSH2, constitutively active Lck mutants lacking the C-terminal regulatory tyrosine or the entire Src homology 2 domain, respectively, required Hsp90 activity to stabilize the mature proteins. Lck synthesized in the absence of Hsp90 activity was degraded within 30–45 min. This unstable Lck was myristoylated normally but did not associate with membranes or CD4, interactions that normally start within minutes of the completion of Lck synthesis. A construct composed of the N-terminal unique domain of Lck fused to green fluorescent protein did not require Hsp90 activity during synthesis. In addition, this protein associated with membranes efficiently in the absence of Hsp90 activity. Together these data suggest that interaction with Hsp90 is necessary for the correct synthesis and subsequent membrane binding of Lck. However, Hsp90 does not appear to play a direct role in Lck membrane, or CD4, association.

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“…In yeast and mammalian cells, the enzymes that prenylate proteins are found in the cytoplasm, but those that mediate proteolysis and carboxylmethylation are localized in the endoplasmic reticulum (reviewed in Magee and Marshall, 1999). Where thioacylation of Ras occurs has not been established, but it has been suggested that thioacylation also occurs early in the secretory pathway (Choy et al, 1999).…”

Section: Plasma Membrane-targeting Via Lipid Modifications and Proteimentioning

confidence: 99%

Dual Lipid Modification Motifs in G_αand G_γSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

Manahan

Patnana

Blumer

et al. 2000

MBoC

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To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide-binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein ␥ subunit (Ste18p) is unusual among G ␥ subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation-or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G ␥ subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G ␤␥ after receptor-stimulated release from G ␣ . The G protein ␣ subunit (Gpa1p) is tandemly modified at its N terminus with amide-and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway. INTRODUCTIONLipid modifications anchor heterotrimeric guanine nucleotide-binding proteins (G proteins) to the inner leaflet of the plasma membrane. G protein ␣ subunits are fatty acylated with amide-linked myristate, thioester-linked palmitate, or both. G protein ␥ subunits are prenylated with either farnesyl or geranylgeranyl moieties through stable thioether linkages. Prenylation of ␥ subunits and myristoylation of ␣ subunits are essential for the function of G proteins. These modifications promote plasma membrane association and facilitate high-affinity protein-protein interactions (reviewed in Wedegaertner et al., 1995). The functional consequences of thioacylation are less well understood. Thioacylation does not appear to be a major determinant of membrane avidity, at least in the presence of G ␤␥ subunits, but may play a role in targeting G ␣ specifically to the plasma membrane (Dunphy et al., 1996;Morales et al., 1998;Fishburn et al., 1999;Huang et al., 1999). In vitro studies have demonstrated the importance of thioester-linked lipid in mediating protein-protein interactions of G ␣ subunits. Thioacylation increases the affinity of G s␣ for G ␤␥ approximately fivefold (Iiri et al., 1996) and negatively regulates the interaction between regulators of G protein signaling and G ␣ subunits (Tu et al., 1997). Thus, thioacylation may impact protein-protein interactions, as well as the subcellular distribution of modified proteins.We have investigated thioacylation of G proteins in the genetically tractable organism Saccharomyces cerevisiae. The pheromone res...

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New Insights into the Interaction of Ras with the Plasma Membrane

Abstract: the S-acylation machinery, the nature of which is still a matter of hot debate (Dunphy and Linder, 1998). On the other hand, the polybasic sequence of K-Ras4B is believed to interact electrostatically with the head groups of anionic phospholipids that are present in the

Cited by 149 publications

References 19 publications

The cholesterol lowering drug lovastatin induces cell death in myeloma plasma cells

The cholesterol lowering drug lovastatin induces cell death in myeloma plasma cells

Hsp90 Is Essential for the Synthesis and Subsequent Membrane Association, But Not the Maintenance, of the Src-Kinase p56^lck

Dual Lipid Modification Motifs in G_αand G_γSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

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New Insights into the Interaction of Ras with the Plasma Membrane

Abstract: the S-acylation machinery, the nature of which is still a matter of hot debate (Dunphy and Linder, 1998). On the other hand, the polybasic sequence of K-Ras4B is believed to interact electrostatically with the head groups of anionic phospholipids that are present in the

Cited by 149 publications

References 19 publications

The cholesterol lowering drug lovastatin induces cell death in myeloma plasma cells

The cholesterol lowering drug lovastatin induces cell death in myeloma plasma cells

Hsp90 Is Essential for the Synthesis and Subsequent Membrane Association, But Not the Maintenance, of the Src-Kinase p56lck

Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

Contact Info

Product

Resources

About

Hsp90 Is Essential for the Synthesis and Subsequent Membrane Association, But Not the Maintenance, of the Src-Kinase p56^lck

Dual Lipid Modification Motifs in G_αand G_γSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae